The radiation-induced bystander effect is a widely described phenomenon which challenges the accepted paradigm of radiation action, but remains poorly understood at a mechanistic level. Genomic instability may be considered to be a driving force in the development of cancer, and ionizing radiations have been shown to be efficient inducers of this latent response. The relationship between these two nontargeted effects has not been established. It is hypothesized that the radiation-induced bystander response and the genomic instability response are mechanistically linked through reactive radical species mediated pathways. Furthermore, we hypothesize that bystander responding cells will show genomic instability at levels equal to that shown by directly radiation damaged cells. Such a finding could augment concerns about the effects of low/very low doses of radiation. We have definitively shown a bystander effect with a precision charged particle microbeam, where nuclear hit cells were knowingly discriminated from non-hit bystander cells. We will determine whether bystander signaling or non-nuclear [cytoplasmic] irradiation can induce genomic instability in mammalian cells. Reactive radical species requirement in bystander and instability response pathways will be tested using specific chemical inhibitors and gene specific siRNA approaches. Frequencies of chromosomal aberrations will be assessed as a function of time. Chromatid/chromosome aberration analysis plus/minus a telomere peptide nucleic acid probe and complete karyotype analyses by multiplex fluorescence in-situ hybridization [m-FISH] in human fibroblast cells, and human chromosome 11 m-BAND analysis in human-hamster hybrid AL cells will be undertaken. Further, we will compare 2D versus 3D responses by studying human bronchial epithelial cells both as 2D monolayers and as 3D airway like tissues, as more closely reflecting cellular behavior in vivo. Four Specific Aims and subsidiary hypotheses will aid in the definition of the mechanistic relationship between the bystander response and genomic instability. Interaction between the Aims of each project will ensure significant progress to fulfillment of the central hypothesis of this P01 in defining the mechanism/s of the radiation-induced bystander effect.